Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.A deno-associated viruses (AAVs) belong to the Parvoviridae family, which is characterized as small animal viruses with linear single-stranded DNA genomes that replicate in the presence of helper virus such as adenovirus (1). AAVs are being evaluated as vectors for human gene therapy (2). The initial characterization of this group of viruses was based on serologic crossreactivity by using complement fixation and neutralizing assays (3). Six distinct serotypes of AAV have been described, of which five were initially isolated as contaminants of adenovirus preparations (4-6). Sequence analysis of selected AAV isolates revealed divergence throughout the genome that is most concentrated in hypervariable regions (HVRs) of the capsid proteins (7-10). Epidemiological data indicate that all known serotypes are endemic to primates, although isolation of clinical isolates has been restricted to AAV2 and AAV3 from anal and throat swabs of human infants and AAV5 from a human condylomatous wart (11)(12)(13)(14). No known clinical sequelae have been associated with AAV infection. Vectors based on replication-defective forms of AAV have been evaluated in preclinical and clinical models of gene therapy (2). Detection and Recovery of AAV Sequences. DNA was extracted and analyzed for the presence of AAV DNA by using a PCR strategy to amplify a 255-bp (15) fragment called the ''signature region'' by using conserved oligonucleotides. To directly amplify a 3.1-kb full-length Cap fragment from NHP tissue and blood DNAs, two other highly conserved regions were identified in AAV genomes for use in PCR amplification of large fragments. A primer within a conserved region located in the middle of the Rep gene was selected (AV1ns, 5Ј-GCTGCGTCA ACTGGACCA AT-GAGAAC-3Ј) in combination with the 3Ј primer located in another conserved region downstream of the Cap gene (AV2cas, 5Ј-CGCAGAGACCAAAGTTCAACTGAAACGA-3Ј) for amplification of full-length cap fragments. The PCR products were Topo-cloned (Invitrogen), and sequence analysis was performed by Qiagengenomics (Qiagengenomics, Seattle) with an accuracy of Ն99.9%. A total of 50 capsid clones were i...
The mechanisms of tolerance in the liver that limit susceptibility to food allergy and that mediate the acceptance of liver transplants even with a complete MHC mismatch remain poorly defined. Here we report that in a model of liver-directed gene transfer cytotoxic T lymphocyte (CTL) responses to non-self antigens are controlled by hepatic regulatory T cells (Tregs) that secrete the immunosuppressive cytokine interleukin (IL)-10 in response to the antigen. In addition, Kupffer cells (KCs), normally thought to initiate immune responses, are rendered tolerogenic in this context. The depletion of KCs results in a complete abrogation of IL-10 production by hepatic Tregs, indicating an interaction between Tregs and KCs in the induction of tolerance. Conclusion Our study suggests that hepatic Tregs together with KCs create a local suppressive microenvironment that prevents the establishment of the CTL response. These mechanisms provide pivotal insights and may prove instrumental in the tolerization toward non-self therapeutic proteins delivered to the liver.
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